Taking a step backward after taking a step forward isn’t a disaster…it’s cha-cha Robert Brault Examination committee : Prof. dr. ir. Paul Van Der Meeren (chairman) Prof. dr. ir. Wim Soetaert (promotor) Dr. ir. Inge Van Bogaert (co-promotor) Prof. dr. ir. Monica Höfte (secretary) Prof. dr. Ibrahim Banat Prof. dr. Bart Devreese Dr. ir. Sofie De Maeseneire Promotors: Prof. dr. ir. Wim Soetaert (promotor) Dr. ir. Inge Van Bogaert (co-promotor) Center of Expertise-Biotechnology and Biocatalysis Department of Biochemical and Microbial Technology Faculty of Bioscience Engineering Ghent University Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. Paul Van Cauwenberge Sophie Roelants was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) ‘Biosurf’ grant number IWT80050 and the European FP7 project ‘Biosurfing’, grant number 289219 Ghent University Faculty of Bioscience Engineering Department of Biochemical and Microbial Technology ir. Sophie Roelants Starmerella bombicola as a platform organism for the production of biobased compounds Thesis submitted for the fulfilment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Academic year: 2012-2013 Titel van het doctoraatsproefschrift in het Nederlands: Starmerella bombicola als platform organisme voor de productie van biogebaseerde moleculen. Cover design and illustrations: Stijn Verweire and Ariane Mol To refer to this thesis: Roelants, S. (2013) Starmerella bombicola as a platform organism for the production of biobased compounds . PhD-thesis, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium. ISBN-number: 978-90-5989-620-8 The author and the promotors give the authorisation to consult and to copy parts of this work for personal use only. Every other use is subject to copy right laws. Permission to reproduce any material contained in this work should be obtained from the author. List of abbreviations LIST OF ABBREVIATIONS at Acetyltransferase gene BS Biosurfactant bp Basepairs CBL Cellobioselipid CFU Colony forming units CMC Critical micelle concentration CSL Corn steep liquor cyp Cytochrome P450 mono oxygenase gene DSP Down stream processing EDTA Ethylene Diamine Tetraacetic Acid ELSD Evaporative Light Scattering Detector FA-OH Hydroxylated fatty acid 5’-FOA 5-fluoroorotic acid gDNA Genomic DNA gt Glycosyltransferase gene HDAC Histon deacetylase HLB Hydrophobic/hydrophilic balance HPLC High Pressure Liquid Chromatography IP Intelectual property kb Kilobases KO Knock Out lip Lipase gene (gene responsible for lactonisation of SLs) LB Luria Bertani (medium) Mb Megabases MELs Mannosylerythritollipids min minutes MTA Material transfer agreement NaCl Sodiumchloride OD Optical density OE overexpression orf Open reading frame PCR Polymerase chain reaction Pgki Promotor of phosphoglycerate kinase Pgapd Promotor of glyceraldehyde 3 phosphate dehydrogenase PHA Polyhydroxyalkanoates RLs rhamnolipids rpm Revolutions per minute sec seconds List of abbreviations SCE Sorbitol Citrate EDTA (buffer) SD Synthetic dextrose medium SM Secondary metabolites SLs sophorolipids TK Tyrosine kinase ura3 orotidine 5-phosphate decarboxylase gene UA Ustilagic acid (cellobioselipid variant) UDP-glucose Uridinediphosphate-glucose (activated sugar) WT Wild type YPD Yeast peptone dextrose medium Outline OUTLINE The organism of interest in this manuscript is the yeast Starmerella bombicola , which is the teleomorph of Candida bombicola (Rosa and Lachance, 1998). At the ‘International Botanical Congress in Melbourne (2011) it was decided that starting from the 1 st of January 2013 one fungus can only have one name, being the name of the teleomorph. Whenever Candida bombicola is thus mentioned in older references, this refers to the same species and in this manuscript and future publications the name of the teleomorph: Starmerella bombicola will be used. This yeast attracted interest for its ability to produce high amounts (above 400 g/L) of a mixture of glycolipid molecules (sophorolipids) with biosurfactant properties. In Chapter I of this manuscript other glycolipids and more generally, biosurfactant producing organisms (both pro- and eukaryotic), their genetic background, the regulatory mechanisms influencing biosynthesis and the biotechnological opportunities these microorganisms present, will be discussed. This will furthermore be discussed in the larger context of secondary metabolite production by eukaryotes as many analogies exist with glycolipid production by yeasts. These sophorolipids produced by Starmerella bombicola are one of the only biosurfactants that are commercialized nowadays. Although numerous publications and patent applications deal with the optimization of fermentation parameters to steer sophorolipid productivity and - composition, very little is known about the physiological role and (molecular) regulation of the production of these secondary metabolites. Moreover, almost no genetic information (besides rDNA sequences) is publically available, let alone the existence of molecular techniques to engineer this industrially important though unconventional yeast. The challenge to do something about this was accepted by researchers at InBio.be and the first achievement was the development of a transformation and selection protocol for S. bombicola (Van Bogaert et al. , 2008b) a few years ago. The work presented in this manuscript was performed in the framework of the national IWT-SBO project ‘BIOSURF’ and European FP7 project ‘BIOSURFING’, which shared a common goal: the transformation of Starmerella bombicola into a platform organism for the production of tailor-made biomolecules. To do so a thorough knowledge of the conditions promoting sophorolipid production in addition to genetic information of the yeast were required. The complete S. bombicola genome was thus sequenced and annotated in the framework of the IWT-SBO project, which led to the discovery of the sophorolipid biosynthetic gene cluster. In the first part of this manuscript (Chapter II and Chapter III) the sophorolipid metabolism of S. bombicola is investigated. In Chapter II the genetic background and possible regulation of Outline sophorolipid production is discussed, whereas in Chapter III one of the possible physiological roles of sophorolipid production, i.e. the creation of an extracellular carbon source, is investigated. The second part of this manuscript (Chapter IV and Chapter V) describes the development of a portfolio of molecular tools for the further genetic engineering of S. bombicola . In Chapter IV the development of a reporter system and a method for the reuse of the auxotrophic ura3 marker is discussed, whereas in Chapter V de development of a qPCR platform is described. The latter will not only serve for the further investigation of sophorolipid metabolism in the wild type organism, but can also be used to determine possible bottlenecks in new strains producing (new-to-nature) biosurfactants. In the third and last part of this manuscript proof of concept is delivered that this yeast can be transformed into a platform organism for the production of interesting biomolecules. In Chapter VI the possibility to fundamentally modify the sophorolipid mixture by applying genetic engineering illustrates the vast biotechnological possibilities this yeast offers. In the seventh and last chapter the conversion of this biosurfactant producer into a bioplastic producer by making use of its efficient SL machinery is described. This second example illustrates that this yeast can not only be used for the production of tailor-made-biosurfactants, but can also serve for the production of completely different biological molecules with interesting perspectives for the biobased economy. Table of contents TABLE OF CONTENTS Acknowledgments………………………………………………………………………………………... List Of Abbreviations ................................................................................................................................ Table Of Contents ..................................................................................................................................... Chapter I. ............................................................................................................................................... 1 Literature Review .................................................................................................................................... 1 I.1. Introduction ......................................................................................................................... 1 I.2. Biosynthesis of biosurfactants and regulation thereof ......................................................... 3 I.2.1. Biosurfactants produced by prokaryotes ..................................................................... 5 I.2.2. Biosurfactants produced by eukaryotes ..................................................................... 16 I.3. Natural role of biosurfactants and other secondary metabolites ........................................ 35 I.3.1. Niche occupation and –protection and survival ........................................................ 36 I.3.2. Competition for and storage of nutrients ................................................................... 38 I.4. Biotechnological opportunities and applications for Biosurfactants ................................. 42 I.4.1. Biotechnological potential of engineered glycolipid producing strains .................... 43 I.5. Production